5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxycytosine (5-caC) were recently identified in mammalian brain and embryonic stem cells as products of the oxidation of 5-methylcytosine (5-mC) by cytosine oxygenases. The biological functions of 5-hmC, 5-fC, and 5-caC are not completely understood; however, several lines of evidence suggest that 5-hmC is involved in epigenetic regulation and DNA demethylation. Iterative oxidation of 5-hmC by cytosine oxygenase enzymes yields 5-fC and 5-caC which are hypothesized to be intermediates in the DNA demethylation process. Several challenges are associated with the identification these biologically modified nucleobases in genomic DNA samples due to their low abundance and temporal fluctuation. Mapping and quantifying 5-mC, 5-hmC, 5-fC, and 5-caC at the DNA level is, therefore, important for unraveling their role in the dynamics of gene expression and regulation.